Intravenous Infusion in the Mouse Using ALZET^® Osmotic Pumps

The development of “knock-out,” transgenic, and new inbred mouse strains, along with increased animal welfare concerns, and higher costs of maintaining larger laboratory animals have encouraged the miniaturization of procedures and techniques. With this trend, has come the need to develop a simple and reliable procedure for the intravenous (IV) infusion of agents to mice. Several methods for chronic IV infusion in the mouse have been proposed, but most have proven too troublesome to be widely accepted as practical methods for drug administration. 1,2,3 The major disadvantage of these protocols is that the mouse must be attached to a tether or swivel during the infusion period, thereby restricting animal movement, preventing group housing of animals, and adding cost and complexity to the procedure. Additionally, these traditional externalized catheters are prone to failure and infection, which present a threat to animal welfare and data integrity.

ALZET osmotic pumps offer researchers a more practical means of chronic IV administration of agents in freely moving, unrestrained mice. Since the pumps are fully implantable, there is no need for external connections, allowing the animals to remain untethered and unrestrained during the entire infusion period. The lack of external connections helps minimize infection and may allow for the group housing of animals. Furthermore, ALZET pumps provide continuous infusion for prolonged periods of time, thereby minimizing stress to the animals that can result from frequent handling and repeated injections.  In addition, the use of our new mouse jugular catheter (part number: 0007700) allows researchers to avoid time consuming modifications to existing catheter tubing.   The proximal end of this new catheter is suited to be used with the ALZET Osmotic Pumps with no additional modifications; while the distal end is a very small 28G polyurethane, designed for IV cannulation in mice.   For more information regarding our new specialized catheters, visit the following webpage of our newly designed site: http://www.alzet.com/products/Alzt_cathrs.php

 

The use of ALZET pumps in mice for IV infusion is discussed in eighteen publications. Other uses of ALZET pumps in mice are described in nearly 1,500 references. Seven of the ten ALZET pump models, ranging in duration from 24 hours to 4 weeks, are appropriate for use in mice. The smallest pump, which has a 100 μl drug reservoir, is suitable for subcutaneous (SC) implantation in mice that weigh at least 10 grams, or intraperitoneal (IP) implantation in mice that weigh at least 20 grams. The 200 μl pumps are suitable for SC implantation in mice that weigh at least 20 grams, and not recommended for IP use. For IV infusion, the pump is implanted SC with the attached catheter leading into the vessel.

An information package containing surgical techniques, references, and tips for IV infusion in mice using ALZET pumps is included. Additionally, the ALZET Technical Information Manual has detailed information on IV surgical techniques in rats, with mention of how to conduct such procedures in mice. Furthermore, a videotape which demonstrates surgical techniques is available free of charge through ALZET Technical Services at 1-800-692-2990.

Mokhtarian A, Meile M-A, Even PC. Physiology & Behavior 1993;54:895-898.
Barr JE, Holmes DB, Ryan LJ, Sharpless SK. Pharmacology Biochemistry & Behavior 1979;11:115-118.

Kelley BM, Bandy A-LE, Middaugh LD. Physiology & Behavior 1997;62(1):163-167.

P8479 Whalen,E.J., Foster,M.W., Matsumoto,A., Ozawa,K., Violin,J.D., Que,L.G., Nelson,C.D., Benhar,M., Keys,J.R., Rockman,H.A., Koch,W.J., Daaka,Y., Lefkowitz,R.J., Stamler,J.S. Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2. Cell 2007; 129(-3-):511-522. >>> Isoproterenol; GSNO; PBS; IV (jugular); Mice; ; Cardiovascular; agent is also known as nitrosoglutathione.

P8224 Menon,J., Soto-Pantoja,D.R., Callahan,M.F., Cline,J.M., Ferrario,C.M., Tallant,E.A., Gallagher,P.E. Angiotensin-(1-7) inhibits growth of human lung adenocarcinoma xenografts in nude mice through a reduction in cyclooxygenase-2. Cancer Research 2007; 67(-6-):2809-2815. >>> Angiotensin (1-7); Saline; heparin; IV (jugular); Mice (nude); 2004; 28 days; Controls received mp w/ vehicle; no stress (see pg. 2810); cancer (lung); peptides; animal info (male, athymic, 2-4 weeks old, 15-20 grams); "During the infusion period, the animals maintained their body weight as well as food and water consumption and showed no evidence of reduced motor function. Additionally, no gross pathological abnormalities were observed in major organs..indicating a lack of toxic side effects." (p. 2810).

P8338 Schaefer,M.B., Ott,J., Mohr,A., Bi,M.H., Grosz,A., Weissmann,N., Ishii,S., Grimminger,F., Seeger,W., Mayer,K. Immunomodulation by n-3- versus n-6-rich lipid emulsions in murine acute lung injury - Role of platelet-activating factor receptor. Critical Care Medicine 2006; 35(-2-):544-554. >>> Saline, physiological; lipoven; omegaven; IV (jugular); Mice; 10 days; Controls received mp w/ saline, physiological; pumps replaced after 7 days of saline infusion; animal info (BALB/C wt, 8-12 weeks old, 18-21 grams, LPS induced acute lung injury and IP inflammation); mp contained an external device tied to the back; lipoven 10% (soybean oil); omegaven 10% (fish oil); lipid emulsion.

P7871 Ohtaki,H., Nakamachi,T., Dohi,K., Aizawa,Y., Takaki,A., Hodoyama,K., Yofu,S., Hashimoto,H., Shintani,N., Baba,A., Kopf,M., Iwakura,Y., Matsuda,K., Arimura,A., Shioda,S. Pituitary adenylate cyclase-activating polypeptide (PACAP) decreases ischemic neuronal cell death in association with IL-6. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2006; 103(-19-):7488-7493. >>> Pituitary adenylate cyclase-activating polypeptide, 6-38; pituitary adenylate cyclase-activating polypeptide 38; pituitary adenylate cyclase-activating polypeptide; Saline; BSA; IV (jugular); Mice; 4, 8 hours; Controls received mp w/ vehicle; peptides; ischemia (cerebral); animal info (BALB/C, IL-6 -/-, +/-, wt); partial or total middle cerebral artery occlusion.

P7135 Benlloch,M., Ortega,A., Ferrer,P., Segarra,R., Obrador,E., Asensi,M., Carretero,J., Estrela,J.M. Acceleration of glutathione efflux and inhibition of gamma-glutamyltranspeptidase sensitize metastatic B16 melanoma cells to endothelium-induced cytotoxicity. Journal of Biological Chemistry 2005; 280(-8-):6950-6959. >>> Acivicin; verapamil; IV; Mice; 2001D; 7 hours; Controls received mp w/ physiological saline; cancer (melanoma).

P6894 Tejeda,M, Gaal,D., Csuka,O., Keri,G.Y. Growth Inhibitory Effect of the Somatostatin Structural Derivative (TT-232) on Leukemia Models. Anticancer Research 2005; 25(--):325-330. >>> TT-232; Acetic acid; sodium acetate; water; mannitol; IV; SC; Mice; 2002; 14, 28 days; Dose-response (p. 328, fig 1); comparison of IP/SC injections vs. SC/IV mp; pumps replaced at day 14 for 28 day group; stability verified, 37 degrees Celsius for over 3 weeks; cancer (leukemia); TT-232 is a novel somatostatin analog; "The IV infusion for 28 days resulted in 82% growth inhibition." (p. 328); "The infusion of TT-232 by ALZET osmotic minipump resulted in 70-80% tumor growth inhibition and 20% tumor free survival." (p. 329); "…serial injections represent significant stress to the animals…" "To reduce and eliminate the above mentioned problem [stress] we used an ALZET osmotic minipump…" "Infusion from inserted ALZET minipumps maintains a constant drug level, resulting in a well defined, consistent pattern of drug exposure throughout the period of drug administration." "These studies suggest that TT-232 is a potent inhibitor of leukemia tumor in vitro and in vivo and suggest infusion treatment as a beneficial application in clinical practice." (p. 330); oligopeptide; enzyme inhibitor (tyrosine kinase); animal info (CBA/ca, immunosuppressed, female).

P7052 Alley,M.C., Hollingshead,M.G., Pacula-Cox,C.M., Wand,W.R., Hartley,J.A., Howard,P.W., Gregson,S.J., Thurston,D.E., Sausville,E.A. SJG-136 (NSC 694501), a novel rationally designed DNA minor groove interstrand cross-linking agent with potent and broad spectrum antitumor activity. Part 2: Efficacy evaluations. Cancer Research 2004; 64(-18-):6700-6706. >>> SJG-136; DMSO; PEG; IV (jugular); Mice (nude); 2001; 5 days; Controls received mp w/ vehicle; comparison of IV injections vs. mp; no stress (see pg. 6706); cancer (melanoma); agent also known as NSC 694501; 50% DMSO; "It is noteworthy that 5-day continuous infusions are highly effective in the LOX IMVI tumor model...continuous infusions were highly active and the lower peak plasma concentrations afforded by them may render infusion a useful mode for SJG-136 delivery in man." (pg. 6704-5).

P6131 Tejeda,M., Gaal,D., Csuka,O., Ullrich,A., Schwab,R., Pap,A., Horvath,A., Keri,G. The antitumor effect of the somatostatin analogue TT-232 depends on th e treatment regimen. Cancer Detection and Prevention 2003; 27(-2-):155-162. >>> TT-232; Acetic acid; sodium acetate; mannitol; SC; IV (jugular); Mice; 2001; 2002; 1,2,4 weeks; Comparison of SC + IV injections vs. mp; 2002 pumps replaced after 14 days; cancer (sarcoma colon, breast lymphoma); somatostatin analog; PE-10 tubing used for IV group; "The infusion treatment with ALZET Osmotic Pumps proved to be superior to both twice daily injections or intravenous injections." (p. 155).

P6035 Ameredes,B.T., Otterbein,L.E., Kohut,L.K., Gligonic,A.L., Calhoun,W.J., Choi,A.M.K. Low-dose carbon monoxide reduces airway hyperresponsiveness in mice. AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY 2003; 285(-6-):L1270-L1276. >>> Rp-8-BrcGMP; Saline; IV (jugular); Mice; 10 days; Rp-8-BrcGMP is an inhibitor of cGMP production; pump model not listed.

P5949 Szende,B., Keri,G. TT-232: a somatostatin structural derivative as a potent antitumor drug candidate. Anti-cancer Drugs 2003; 14(-8-):585-588. >>> TT-232; SC; IV; Mice (SCID); 2002; 14 days; Comparison of SC injections vs. mp; cancer (sarcoma, lymphoid, colon, breast); peptides; TT-232 is a somatostatin analog; "the infusion treatment using implanted ALZET-type osmotic mini-pumps proved to be superior to both S.C. and I.V. infusion applied twice a day for 2 weeks." (p. 586).

P6973 Arap,W, Haedicke,M, Bernasconi,M, Kain,R, Rajotte,D  PNAS 2002; 99(-3-):1527-1531. >>> SMSIARL, phage; IV; Mice; ; Comparison of pellets vs. mp; peptides; cancer.

P5554 Bowler,R.P., Sheng,H.X., Enghild,J.J., Pearlstein,R.D., Warner,D.S., Crapo,J.D. A catalytic antioxidant (AEOL 10150) attenuates expression of inflammatory genes in stroke. Free Radical Biology and Medicine 2002; 33(-8-):1141-1152. >>> AEOL 10150; PBS; IV (jugular); Mice; 1003D; 6 hours; Controls received mp w/ vehicle; AEOL 10150 is a catalytic antioxidant metalloporphyrin; ischemia (cerebral).

P5314 Sheng,H.X., Enghild,J.J., Bowler,R., Patel,M., Batinic-Haberle,I., Calvi,C.L., Day,B.J., Pearlstein,R.D., Crapo,J.D., Warner,D.S. Effects of metalloporphyrin catalytic antioxidants in experimental brain ischemia. Free Radical Biology and Medicine 2002; 33(-7-):947-961. >>> AEOL 10150; PBS; IV (jugular); Mice; 1003D; 3 days; Controls received mp w/ vehicle; plasma levels of AEOL 10150 determined by HPLC; ischemia (cerebral).

P8575 Pavco,P.A., Bouhana,K.S., Gallegos,A.M., Agrawal,A., Blanchard,K.S., Grimm,S.L., Jensen,K.L., Andrews,L.E., Wincott,F.E., Pitot,P.A. Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors. Clin Cancer Res 2000; 6(-5-):2094-2103. >>> Ribozyme, anti-Flt-1; ribozyme, anti-KDR; ribozyme, attenuated; Saline; IV (jugular); SC; Mice; mice (nude); 2002; 14,28 days; Controls received mp w/ vehicle or attenuated control; dose-response (fig. 2); pumps replaced after 18 days; stress/adverse reaction (see pg. 2096);<10% of animals removed from study due to catheter failure or animal not surviving pump implantation; half-life (p. 2100) IV and SC similar; cancer (lung carcinoma, colorectal carcinoma, liver metastasis); animal info (female, C57BL/6, 6-8 weeks old, male, nude, 6-8 weeks old); antiangiogenic, 80% bioavailable after SC administration; targets VEGF receptor mRNA; anti-Flt-1 ribozyme also known as angiozyme.

P5249 Sharkey,E.M., O'Neill,H.B., Kavarana,M.J., Wang,H., Creighton,D.J., Sentz,D.L., Eiseman,J.L. Pharmacokinetics and antitumor properties in tumor-bearing mice of an enediol analogue inhibitor of glyoxalase I. Cancer Chemother.Pharmacol 2000; 46(-2-):156-166. >>> Glutathione, S-(N-p-chlorophenyl-N-hydroxycarbamoyl); Cyclodextrin, B-; IV (jugular); Mice; 2002; 14 days; Comparison of IV injections vs. mp; enzyme inhibitor; 20% hydroxypropyl-beta-cyclodextrin used; CHG (Et)2 is an enediol analog inhibitor of glycoxalase in its ester prodrug form; colon adenocarcinoma and prostate cancer.

P4485 Tejeda,M, Gaal,D., Schwab,R.E, Pap,A, Szúts,T, Keri,GY. Influence of various administration routes on the antitumor efficacy of TT-232, a novel somatostatin analog.. Anticancer Res. 2000; 20(--):1023-1028. >>> TT-232; Acetic acid; Water, distilled; Sodium acetate; Marmite;; SC; IV; Mice; 2001; 2002; 1, 2 weeks; comparison of IV + SC injections vs. mp infusion; cancer; peptides; "continuous infusion using an implantable minipump proved to be the most effective route of treatment in both SC or IV administration against S-180 sarcoma." (P. 1026).

P5566 Dono,R., Texido,G., Dussel,R., Ehmke,H., Zeller,R. Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice. EMBO J. 1998/8/3; 17(-15-):4213-4225. >>> Angiotensin II; IV (jugular); Mice; 1007D; 6 days; Cardiovascular; peptides.

P3363 Tu,Y., Stepkowski,S.M., Chou,T.-C., Kahan,B.D. The synergistic effects of cyclosporine, sirolimus, and brequinar on heart allograft survival in mice. Transplantation 1995; 59(-2-):177-183. >>> Cyclosporin A; Rapamycin; Cremophor; Dimethylacetamide, N, N-; Tween 80; PEG 400; IV (jugular); mice; 1007D; 7, 14 days; pumps replaced after 7 days; immunology; CSA and rapamycin given singly and via same pump.

P2048 Stepkowski,S.M., Tu,Y., Condon,T.P., Bennett,C.F. Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities. J.Immunol. 1994; 153(--):5336-5346. >>> Oligodeoxynucleotide, phosphorothioate antisense; Cyclosporin A; Rapamycin; Oligodeoxynucleotide, scrambled; Cremophor; Tween 80; Dimethylacetamide, N, N-; PEG 400; IV (jugular); mice; 7,14 days; oligo of interest was IP-3082; controls received no treatment, unrelated oligo IP-1082, or scrambled oligo IP-4189; immunology; "...this study reports the first example of pharmacologic activity of an antisense PS-oligo by i.v. systemic administration in a model of a complex inflammatory process".

P2096 Redwood,S.M., Liu,B.C.S., Weiss,R.E., Hodge,D.E., Droller,M.J. Abrogation of the invasion of human bladder tumor cells by using protease inhibitor(s). Cancer 1992; 69(-5-):1212-1219. >>> E-64; PBS; IV (jugular); mice (nude); no duration posted; cancer; immunology; enzyme inhibitor (cysteine protease).

P1464 Nesbitt,T., Davidai,G.A., Drezner,M.K. Abnormal adenosine 3'.5'-monophosphate stimulation of renal 1,25-dihydroxyvitamin D production in Hyp mice: evidence that 25-hydroxyvitamin d-1a-hydroxylase dysfunction results from aberrant intracellular function. Endocrinology 1989; 124(--):1184-1189. >>> Cyclic AMP, N-6-monobutyryl; Water; IV (jugular); mice; 2001; 24 hours; no comment posted.

P1160 Naito,K., Pellis,N.R., Kahan,B.D. Effect of continuous administration of interleukin 2 on active specific chemoimmunotherapy with extracted tumor-specific transplantation antigen and cyclophosphamide. Cancer Res. 1988; 48(--):101-108. >>> Interleukin-2; IP; IV; mice; 1702; 2001; 2002; 2ML2; 10 days; mp connected to catheter; half-life; 2 doses infused; IL-2 infused to potentiate effects of TSTA/CY inject.; comparison of bolus inject. vs. inject. vs. mp infusion; tissue perfusion (spleen); cancer/immunology; peptides; human IL-2 used.

P0963 Ishida,K., Yoshimura,K. Eosinophil responses of permissive and nonpermissive hosts to the young adult worms of Angiostrongylus cantonensis. Z.Parasitenkd 1986; 72(--):661-671. >>> Worm extract -A.cantonensis; Worm secretions -A.cantonensis; IP; IV (jugular); guinea-pig; mice; rat; 2001; 7 days; mice implanted w/mp ip other animals infused iv; host-parasite response; eosinophilia determination; comparison of iv injections vs. mp infusion.

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SURGICAL TECHNIQUES FOR MOUSE INTRAVENOUS INFUSION

The following pages include a brief listing of references on suggested surgical techniques and general laboratory practices for intravenous cannulation in mice. A short summary of the cannulation technique used by each group follows the citation.

If you have any questions on this methods summary or on the use of ALZET osmotic pumps in general, please call ALZET Technical Services at 1-800-692-2990.

 

General tips for mouse IV cannulation:

• Strict aseptic technique should be followed. When working with immunocompromissed animals, the surgery should be performed in a sterile hood.
• The external jugular vein is the best choice for IV cannulation in mice.
• The IV cannulation procedure is an extrapolation from the procedure described for the rat, except that the small size of the animal requires smaller instruments (ophthalmologic ones are best) and the necessity to operate under a magnification glass to visualize the fine operative details.
• A high quality microsurgical apparatus and basic microsurgical skills are invaluable. Dissecting scopes, surgical loops, or magnifying lights are less acceptable alternatives since they may not magnify the surgical field to the required level .
• Use good quality surgical instruments and make sure that they are small enough for the task. Specialized instruments such as vessel dilators and catheter introducers are recommended.
• The small size of the vessels in the mouse requires the use of small diameter catheter tubing. The universal flow moderator of the pumps will accept any tubing that will grip a No. 21 gauge needle (e.g., PE-60 tubing). There are two ways of achieving the necessary step-down in catheter size.
• Polyethylene tubing can easily be drawn out over a heat source to a very small size. Hold the PE tubing about 6" above a low flame, and rotate the tubing while moving it back and forth across the flame, pulling steadily. After the tubing has been drawn out, attach a water-filled syringe connected to a 21-gauge needle to the end that has not been exposed to the heat source. Apply pressure on the plunger, and using scissors cut off the attenuated end in successive pieces until water flows out. This is the smallest diameter you can reasonably obtain with this procedure.
• We have successfully obtained open tips no larger than 4X the diameter of a red blood cell by this means, while the base end of the catheter retained its original geometry to fit on a 21-gauge needle.
• Another method for using very small bore catheters with ALZET pumps is to glue PE-10 inside PE-60 tubing. Be sure to test for leakage at this junction before implanting the catheters. PE-10 tubing is commercially available from Fisher Scientific, P.O. Box 58056, Santa Clara, CA 95050; phone: (800) 766-7000; fax: (408) 727-4905.

 

General pump considerations:

• Choose the appropriate pump for the desired delivery rate and duration: 100 and 200 microliter ALZET pumps are suitable for use in mice. The smallest pump, which has a 100 m l drug reservoir, is suitable for subcutaneous (SC) implantation in mice that weigh at least 10 grams, or intraperitoneal (IP) implantation in mice that weigh at least 20 grams. The 200 m l pumps are suitable for SC implantation in mice that weigh at least 20 grams, and they are not recommended for IP use.   For IV infusion, the pump is implanted SC with the attached catheter leading into the vessel.
• Attach catheter tubing to the short portion of the flow moderator which is exposed after removing the cap on the 200 microliter pumps. When using osmotic pump models 1003D, 1007D, or 1002 with a catheter, a special step is required. With these models only, you should remove the white plastic flange from the flow moderator using a pair of scissors or pliers. In removing the flange, be careful not to bend or crush the stainless steel tube. After the white plastic flange has been removed, attach the catheter tubing to one end of the flow moderator (insert the catheter over approximately 2-3 mm of the flow moderator) The flow moderator can then be inserted into the pump.
• Fill and prime pump under sterile conditions. Prime the pumps overnight in sterile saline at 37 o C (the 2004 Model needs to be primed for 40 hours).   This should prevent any retrograde diffusion back into the pump and drug reservoir.   This priming time is needed to properly hydrate the osmotic sleeve to ensure the continuous delivery of your agent. This step is also necessary to ensure catheter patency.
• Weigh the pump before and after filling to verify that the pump has been filled completely with drug solution. Air bubbles trapped within the body of the pump may result in unpredictable pumping rate fluctuations.

 

Myrback M. and Urquhart J., personal communication. 1994

The following tips have been drawn from the personal surgical experiences of Mick Myrback and Dr. John Urquhart, two of ALZA's experts in animal surgery.   These suggestions are intended to provide technical advice to investigators pursuing intravenous drug infusion in mice using ALZET Osmotic Pumps.

• Practice and proper surgical technique are paramount in successful IV cannulation in mice.   It is best to practice cannulating on both external jugulars until one feels comfortable with the procedure.
• The issue of catheter size may be judged simply by looking at the animal to estimate how big the vein appears to be. The catheter should not be markedly smaller than the vein, but approximately three-fourths the diameter of the undisturbed vessel.
• Mick Myrback prefers to use medical grade vinyl tubing size V-3, as he does not like to use polyethylene tubing for these kind of delicate applications. Vinyl catheter tubing may be purchased from DURECT Corporation. at (877) 922-5938 or (408) 865-1406 ( fax ).
• Dr. Urquhart suggests putting a slight lip on the catheter by holding a hot cautery close to the catheter tip to flare it slightly.
• Perform the surgery while using some kind of magnification headpiece. For example, one may use a headpiece similar to a jeweler's headband with a fixed 10-power lens.   This is particularly helpful since structures in the mouse are very small.   In addition, Dr. Urquhart usually is only about four inches away from the animal as he performs the surgery.
• Make sure to tease away any connective tissue around the vein. However, do not manipulate the vein or touch it more than absolutely necessary since excessive manipulation will cause the vessel to shrink up.
• Correct exposure and excision of the vein are very important.   If one cuts the vessel properly, it will collapse, looking like a flat, round hole with intima on the other side.   If one picks up the edge of this hole at about 2 o'clock with the right forceps, the hole will yawn open and one can then slide the catheter in easily.

Foster HL, Small JD and Fox JD. The Mouse in Biomedical Research. 1983;Vol III: 419-420.

• The authors suggest the following procedures for performing microsurgery in mice:
• Use ophthalmologic or watchmaker's instruments, and ophthalmologic suture
• Use a binocular dissecting microscope with good depth of field
• Perform surgery with at least a 5-inch working distance
• A basic surgical pack for abdominal surgery in mice should include the following:
• A pair of microdissecting thumb forceps and a pair of sharp-sharp microdissecting scissors that are used to incise the skin.
• A pair of microdissecting thumb forceps and a pair of sharp-sharp or blunt-sharp microdissecting scissors that are used to incise subcutaneous tissues and the abdominal wall.
• One or two pairs of microdissecting thumb forceps, one or two pairs of curved or straight watchmaker's forceps and a pair of cataract scissors for manipulation and incision of abdominal viscera.
• Small pieces of Gelfoam (absorbent surgical sponge) or flat-bladed toothpicks wrapped with surgical cotton to control hemorrhage.
• Size 4-0 to 6-0 suture for ligature of vessels and closure of skin incisions. Wound clips may also be used to suture the skin. If wound clips are used, care must be taken to evert, not to invert, the edges of the skin. Ophthalmologic sutures with pre-attached needles save time and reduce tissue trauma.
• Baby Derf, Castroviejo, or Noyes needle holder.
• Paper clip retractors or eyelid retractors for retraction of abdominal walls or viscera.
• Sterile surgical drapes.

Other notes:

• Mouse skin and abdominal muscles are thin and fragile compared to other species and cut edges tend to curl under during the suturing process. In the author's experience, interrupted horizontal mattress sutures are preferred suture patterns for mouse skin or abdominal muscles because they minimize inversion of the skin or peritoneal edges.

 

Mokhtarian A, Meile M-A and Even PC. Chronic Vascular Catheterization in the Mouse. Physiology & Behavior 1993;54:895-898.

• Silastic catheter was used to cannulate the right external jugular vein.
• The mouse was relaxed by IP injection of a myorelaxant (xylazine, 0.05 ml of 2% solution, Rompun Bayer)
• Five minutes later the mouse was anesthetized with 15 mg/100 g of ketamine chloral hydrate (Imalgen 500).
• To limit the chance of infection, the surgery was performed using a face mask, and the operative field, instruments and catheter were washed with a disinfectant solution (Sporicidine, Peters France).
• The catheter was filled with heparinized saline (2500 U heparin/250 ml saline).
• The surgical procedure is an extrapolation to the mouse of the technique described for the rat, except that the small size of the animal requires smaller instruments (ophthalmologic ones are best) and the necessity to operate under magnification.
• A longitudinal incision was made in the skin just over the spot were the anterior jugular, acromiodeltoid, and cephalic veins join together; then, the connective tissue surrounding this spot was carefully removed.
• Two thin threads of polyester were passed under the jugular vein below the level of the junction and separated by about 3 mm.
• The cephalic thread was tied to prevent bleeding.
• A small incision was made just below the ligature, and the cannula was pushed into the lumen (11.5 mm for a 20 g mouse, 12.7 mm for a 30 g mouse, and 14 mm for a 40 g mouse).
• A few drops of blood were withdrawn to confirm correct catheter placement into the cavum.
• The catheter was then fixed with the thread previously used to tie the jugular vein.
• Backward blood flow from the heart was prevented by loosely tying the jugular vein around the catheter with a second thread.
• As a prophylactic measure, the mouse was injected SC with 0.5 mg of gentamicine in 0.05-ml volume.
• The mouse was kept in a warm place until fully recovered from anesthesia.

Brown DF and Burr RE. Customized Vascular Catheters for Rodents. Laboratory Animal Science 1985;35(5):515-516.

• Polyurethane tubing (Micro-renathane â , Braintree Scientific, Inc.) was chosen for vascular catheterization for the following reasons:
  • It is non-toxic, non-thrombogenic, and chemically stable.
  • It is rigid yet pliable and can be sterilized.
  • It can be modified into various shapes. It can be easily customized to fit blood vessels in small rodents without compromising catheter biofunctionality.
• Polyurethane tubing with I.D. of 0.64 mm and O.D. of 1.02 mm was used. To modify the size of the catheter, a loop of tubing was dipped into a beaker of hot (160 o C) peanut oil for two to five seconds. Immediately upon removal from the oil, the tubing was pulled, resulting in a taper. A faster pull produced a more abrupt taper. The distance the tubing was pulled determined the tubing diameter. Longer pulling distances provided finer tubes with miniature diameters.
• The tubing remained patent unless pressed, crimped or melted.
• The rigidity of the tubing was fairly well maintained following the heat treatment.
• Tubing end flares were made by cutting tapered tubes on a bevel at the oil-air interface. Alternatively, the tubing was flared by dipping the end into hot oil.
• Catheters were inserted into carotid arteries or jugular veins up to three centimeters and filled with heparin (1000 U/ml).   Note: Polyurethane tubing is gas permeable. This feature promotes evaporation of the solution (heparin) from the catheter portion exposed to air, thereby promoting clot formation. Clotting was prevented using a 9:1 mixture of glycerin and heparin (1000 units/ml). By filling catheters with this solution, catheter patency was maintained for over three weeks.
• The tubing was sutured to the muscle.

 

Barr JE, Holmes DB, Ryan LJ and Sharpless SK. Techniques for the Chronic Cannulation of the Jugular Vein in Mice. Pharmacology Biochemistry & Behavior 1979;11:115-118.

• Silicone rubber tubing was used to allow flexibility.
• Adult mice were anesthetized with 280-mg/kg chloral hydrate and 140-mg/kg ketamine in saline vehicle.
• The incision, location of vein, insertion of the cannula and closing of the wound follow the procedure developed for the rat.
• The author suggests the use of a 27 gauge needle or fine microdissection scissors to make the insertion hole in the vein.
• The use of a dissection microscope was extremely helpful in visualizing the finer operative details.
• Silicone rubber tubing (I.D. 0.30 mm, O.D. 0.64 mm) was attached to silicone rubber tubing (I.D. 0.51 mm, O.D. 0.94 mm). Insertion was accomplished by allowing the two tubing types to expand in either chloroform or trichloroethane and inserting the smaller tubing 4-mm into the larger tubing. The junction between the two tubing must be made some distance above the point of insertion to avoid clotting. 1.2 mm of heat shrinkable polyolefin tubing was used as a tie-down structure and to strengthen the catheter junction.
• Tubing may be cut at a bevel to facilitate implantation, but bevels of more than 30 o off the transverse plane are not recommended, as they tend to puncture the vein.
• Recommended intra-jugular cannula insertion length: 10-14 mm for mice weighing from 18 to 30 g.

Kelley BM, Bandy A-LE and Middaugh LD. A Novel Chronic and Detachable Indwelling Jugular Catheterization Procedure for Mice. Physiology & Behavior 1997;62(1):163-167.

• At the time of surgery, the mice were approximately 12-13 weeks old and had an average weight of 30 g.
• Animals were anesthetized with an IP injection of a cocktail mixture of ketamine hydrochloride (10 mg/kg in 0.01 ml/g volume) and xylazine hydrochloride (0.2 mg/kg in 0.003 ml/g volume).
• Catheter construction: the intravenous part of the catheter had a 10-mm length of Silastic tubing (I.D. 0.011 in.; O.D. 0.024 in.) beveled at a 45 o angle to facilitate insertion into the jugular vein. This tubing was inserted 3 mm into another gauge of Silastic tubing (I.D. 0.020 in.; O.D. 0.037 in.). The latter catheter was attached to polyethylene tygon tubing (Tygon, I.D. 0.031 in.; O.D. 0.093 in.). The catheter junctions were reinforced with Super Glue Gel ä .
• Mice were placed dorsally on a warm sterile cloth during the procedure.
• An 8-10 mm ventral paramedian skin incision was made superficial to the carotid artery and parallel to the trachea.
• The right, external jugular vein was made accessible using blunt dissection of surrounding tissue and was kept moist with sterile water to prevent collapse.
• Two ligatures were placed at least 2 mm apart, both distal to the origin of the axillary vein. After the rostral ligature was tightened, the right jugular was venotomized between the two ligatures with a 27-gauge needle; a dissection microscope was essential during this part of the surgery.
• The tubing was filled with heparinized saline (20 mg/ml) and was inserted 7-10 mm into the jugular vein. After the catheter was in place, a small amount of blood was drawn into the catheter and it was then flushed with heparinized saline to ensure catheter patency.
• The catheter was secured and the vein was occluded by tightening the proximal ligature and by tying additional knots above and below the silastic collar.
• The distal part of the catheter was directed subcutaneously.
• Care should be taken to ensure that the catheter does not become twisted.
• Tissue adhesive (Vetbond TM No.1469, 3M Animal Care Products) was used to close the ventral incision. Tissue adhesive was found to be superior to staples or sutures in closing the wounds because it was more rapid, did not induce further injury, completely sealed the incisions, acted as a barrier to keep foreign matter from entering the wound, and was shed as healing occurred.
• Triple antibiotic ointment was applied to the incision site.

 

Nesbitt T, Davidai GA and Drezner MK.   Abnormal Adenosine 3'.5'-Monophosphate Stimulation of Renal 1,25-Dihydroxyvitamin D Production in Hyp Hydrolase Dysfunction results from Aberrant Intracellular function. Endocrinology 1986;124(3):1184-1189.

• ALZET osmotic pump model 2001 was used for continuous IV administration of N6-monobutyryl cAMP in C57BL/6J mice.
• Pump reservoirs, as well as 8 inch pieces of polyethylene tubing (PE-10, Clay Adams), were attached to the portal ends of the pumps and filled with solution.
• Pumps were incubated in physiological saline for 18 hours to permit flow rate equilibrium.
• Mice were anesthetized with sodium pentobarbital (65 mg/kg, IP).
• The left jugular vein was exposed, the proximal end was ligated, and two 5-0 silk sutures (Ethicon Inc.) were pre-placed around the distal end of the vein. The free end of the polyethylene tube was inserted into the vein through a small nick made between the two pre-placed ligatures. Both ligatures around the distal vein were tied, and the free ends of the ligatures around the proximal portion of the vein were tied around the catheter.
• Subsequently, the catheter was looped around, and two stay sutures (one to the digastricus muscle under the chin and the other to the pectoral muscle over the chest) were placed to further secure the catheter and prevent its dislodging from the vein.
• The skin incision was closed with simple interrupted sutures of 5-0 silk.
• The catheter was tunneled subcutaneously over the rib cage, and the pump inserted SC over the dorsal lumbar area after making a second skin incision to manipulate the pump.
• Finally, the skin incisions were closed with simple interrupted sutures of 5-0 silk.
• Pumps were implanted 12, 18, and 24 hours before exsanguination